This application offers an innovative solution for improving the Nation's capability in diagnostics for biodefense. Currently Sentinel Labs of the Laboratory Response Network (LRN) performs routine tests that might rule out a biological warfare agent but often cannot perform follow-up tests because these might be too hazardous for BSL2 facilities or are too complex. These more complex and biohazardous tests must be performed at Reference or National Labs of the LRN. IQuum has developed a Lab-in-a-Tube (Liat(tm)) technology which could be used at Sentinel Labs to detect Bioterror agents within 1 hour of patient presentation with symptoms associated with a possible bioterror attack. The Liat system enables minimally trained healthcare practitioners to perform a complex nucleic acid test using whole blood as a sample input. As the Liat system uses a closed tube to perform all sample processing operations, tissue processing and extraction of nucleic acids of agents that require BSL3 and BSL4 confinement for these operations could be performed in BSL2 facilities. We propose to develop a rapid reverse transcriptase polymerase chain reaction (RT-PCR) assay on the Liat system for the diagnosis of bacterial and rickettsial bioterror agents using the 16s rRNA gene. Because each bacterium contains 1,000 to 10,000 copies of rRNA, the RT-PCR assay is expected to have highly sensitive bacteremia detection. By exploiting sequence variation between the variable domains of the 16S rRNA of different strains, it will be possible to further characterize the amplification products to identify the putative causative agent(s) of a given pathology. In addition, we will use a second test targeting the groE/OmpB/gltA genes to further refine the identification of pathogens. IQuum will collaborate with Tufts University to characterize the 16s rRNA assays as well as with the Naval Medical Research Center (NMRC) to characterize the groEL/OmpB/gltA assays in pre-clinical trials using simulated clinical samples. [unreadable] [unreadable] [unreadable]